Name: isolation, characterization and functional examination of the gingival immune cell network
Uploaded: 2016-02-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

Alla experimentella procedurer som beskrivs i detta protokoll följde nödvändiga riktlinjer och godkändes av Institutional Animal Care och användning kommittén, NIDCR / NIH. 1. Förbered i förväg <ol><li> Förbereda Komplett medium: RPMI kompletterat med 2 mM L-glutamin, 100 enheter / ml penicillin, 100 pg / ml streptomycin och 10% FBS. </li><li> Förbered DNas media: 50 ml RPMI-medium kompletterat med 7,5 mikrogram DNas (Se frisk, hålla på is hela tiden). </li><li> Förbered Kollagenas-DNas media: 5 ...

<ol>
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Den nuvarande tekniken med framgång ger ett stort antal immunceller (från en enda mus) lämplig, inte bara för fenotypisk karakterisering, men även för funktionella studier ex vivo. En annan grupp hade tidigare publicerat ett protokoll för att isolera och karaktärisera murin tandköttsimmunceller och introducerade värdet av att använda multi flödescytometri i studiet av parodontit i djurmodeller 9. Efter avsevärd felsökning nyckeln ändring i detta protokoll var att inkludera dissektion a...

Tandköttsvävnader omger humana och murina tänder och utsätts ständigt för komplexa biofilm av tanden 1. Immuncellnätverket polistandkötts barriären är viktigt att upprätthålla vävnadsintegritet, se homeostas med den lokala kommen mikrober och samtidigt ger effektiv immunitet mot patogena utmaning 2. För att uppnå homeostas, är immunsystemet noggrant anpassade till tandkötts miljön skapar en högt specialiserad immunceller nätverk, men ändå liten detalj är känd av tandköttsimmuncellpopulationer och deras roll för att upprätthålla vävnads immunitet 2. När immun homeostas störs på tandköttet, antingen genom ökad värd känslighet och / eller närvaro av dysbiotic mikrobiella samhällen, ett inflammatoriskt tillstånd, uppstår parodontit 3 4. Parodontit är en vanlig inflammatorisk sjukdom, vilket leder till förlust av tand supporting strukturer. I sina allvarliga former det ses hos cirka 10% av befolkningen 5. Dissekera nyckelfaktorer som är involverade i parodontit känslighet och progression har visat sig svårt sex. Däremot har djurmodeller varit extremt användbar för att förstå mekanismerna bakom parodontit initiering och progression 7. Modeller kan användas för att definiera de viktigaste cellpopulationer och molekylära mediatorer som är avgörande för att upprätthålla immun homeostas och driva utvecklingen av parodontit. En sådan insikt kommer att förändra vår förståelse av tandköttet specifik reglering av immun homeostas och ytterligare vår nuvarande förståelse av sjukdomen patogenes.

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isolation, characterization and functional examination of the gingival immune cell network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

För att åskådliggöra tillämpning av protokollet visar vi representativa resultat undersöker immunceller nätverk i tandköttet hos möss med och utan parodontit (WT kontra LFA - / -, figur 2A - C). Representativa FACS tomter visar levande CD45 + hematopoetiska celler i tandköttet (Figur 2A, 2C). Isolering och bearbetning av immunceller med detta protokoll ger tillräckligt många celler för ...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Vi har etablerat en teknik för isolering, fenotypiska karaktärisering och funktionell analys av immunceller från mus tandkött.

Isolering, karakterisering och funktionella Undersökning av tandkötts Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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